Angiogenin Expression in Plants

ABSTRACT

The present invention relates to plant-produced angiogenins, to related plant cells, plant calli, plants, seeds and other plant parts and products derived therefrom and to uses of plant-produced angiogenins. The present invention also relates to expression of angiogenin genes in plants and to related nucleic acids, constructs and methods.

FIELD OF THE INVENTION

The present invention relates to plant-produced angiogenins, to relatedplant cells, plant calli, plants, seeds and other plant parts andproducts derived therefrom and to uses of plant-produced angiogenins.

The present invention also relates to expression of angiogenin genes inplants and to related nucleic acids, constructs and methods.

BACKGROUND OF THE INVENTION

Angiogenin, encoded by the ANG gene, is a member of the ribonuclease(RNase) superfamily. Angiogenin (also known as RNase5) is a 14 kDa,non-glycosylated secreted ribonuclease polypeptide. Angiogenin is knownto regulate the formation of new blood vessels through a process calledangiogenesis and is known to regulate neuron survival with functionalmutations in the protein a cause of the neuromuscular disorderamyotrophic lateral sclerosis (ALS).

During angiogenisis, the angiogenin protein binds to receptors on thesurface of endothelial cells and smooth muscle cells and undergoesnuclear translocation where it stimulates the production of ribosomalRNA (rRNA) which is required for the growth and division of cells forcapillary formation. Angiogenesis associated with exercise causescapillary growth that allows for greater nutrient and oxygen delivery tomuscle tissue.

In our co-pending application PCT/AU2009/000603 we demonstrated thatangiogenin increases muscle cell growth and differentiation in vitro,and significantly alleviates the potent inhibitory effects of myostatinon myoblasts. Angiogenin is enriched in colostrum and milk, secretionswhich evolved to promote health, growth and development of sucklingmammals. When added to the feed of mice, angiogenin purified from bovinemilk increased exercising muscle growth by 50% over a 4 week period. Wedemonstrated that angiogenin is bioavailable when administered orally inour co-pending application PCT/AU2009/000602.

Angiogenin has also been shown to possess a number of other activities.These include the ability to remove skin defects such as pigmentedspots, modulation of immune responses, protection of polymorphonuclearleukocytes from spontaneous degradation, and microbicidal activityagainst systemic bacterial and fungal pathogens. Angiogenin also appearsto be required for effective activity of growth factors such as VEGF,EGF and FGF. In addition, functional mutations in the angiogenin proteincause the neuromuscular disorder amyotrophic lateral sclerosis (ALS).

Angiogenin may have numerous applications, including applications inmedicine, dietary foodstuff supplements and cosmetics. However, the useof angiogenin in such applications requires an efficient process for thepreparation of the protein on a commercial scale from an appropriatesource.

Angiogenin is readily available in bovine milk, however its use as asource of angiogenin is not favoured as angiogenin is only present inbovine milk at a low level. Also, certain proteins, such as caseins, andmilk whey proteins such as immunoglobulin, lactoferrin andlactoperoxidase present in milk mask angiogenin, hindering itspurification.

Reference to any prior art in the specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any otherjurisdiction or that this prior art could reasonably be expected to beascertained, understood and regarded as relevant by a person skilled inthe art.

It is an object of the present invention to overcome, or at leastalleviate, one or more of the difficulties or deficiencies associatedwith the prior art.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a plant cell, plantcallus, plant, seed or other plant part including an angiogenin gene ora functionally active fragment or variant thereof and/or an angiogeninpolypeptide.

In a second aspect, the present invention provides methods of usingplant cells, plant calli, plants, seeds or other plant parts includingan angiogenin, for example as feed stock or for human consumption.

In a further aspect, the present invention provides a plant-producedangiogenin.

In a further aspect, the present invention provides a feedstock, foodsupplement or veterinary product including a plant-produced angiogenin.

In a further aspect, the present invention provides a food, beverage,food supplement, nutraceutical or pharmaceutical including aplant-produced angiogenin.

In a further aspect the present invention provides a method forproducing a transformed plant cell expressing an angiogenin gene.

In a further aspect, the present invention provides methods of isolatingangiogenin from transformed plant cells.

In a further aspect, the present invention provides methods ofregenerating transformed plant calli, plants, seeds or other plant partsfrom transformed plant cells.

In a still further aspect, the present invention provides methods ofisolating angiogenin from transformed plant calli, plants, seeds orother plant parts.

In a still further aspect, the present invention provides methods ofenhancing expression, activity or isolation of angiogenin in plants,said methods comprising co-expressing angiogenin with a mediator ormodulator of angiogenin activity.

In a still further aspect, the present invention provides an artificialconstruct including an angiogenin gene, said construct enablingexpression of said angiogenin gene in a plant cell.

In a still further aspect, the present invention provides artificialconstructs or chimeric sequences comprising an angiogenin gene and agene encoding a mediator or modulator of angiogenin activity.

In a still further aspect, the present invention provides a chimericsequence comprising an angiogenin gene and a plant signal peptide.

In a still further aspect, the present invention provides an angiogeningene with codon usage adapted for plants to enable expression of saidangiogenin gene in a plant cell.

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

As used herein, except where the context requires otherwise, thesingular forms “a”, “an” and “the” include plural aspects.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1. Nucleotide sequence of the Bos taurus angiogenin, ribonuclease,RNase A family, 5 (ANG) (SEQ ID NO: 1). NCBI Accession NM_(—)001078144.The 72 bp signal peptide sequence identified by NCBI is in bold andunderlined.

FIG. 2. Amino acid sequence of the Bos taurus angiogenin, ribonuclease,RNase A family, 5 (ANG) (SEQ ID NO: 2). NCBI Accession NP_(—)001071612.The 24 aa signal sequence identified by NCBI is in bold and underlined.The angiogenin receptor binding domain is highlighted in

and the active site residues are highlighted in

. The Asp (D) amino acid highlighted in bold and underlined is apossible site for mutation to enhance angiogenin activity.

FIG. 3. Nucleotide sequence of the Bos taurus angiogenin, ribonuclease,RNase A family, 5 (ANG) (SEQ ID NO: 3) modified for plant codon bias asdefined by Murray et al., (1989). No changes in amino acid sequence tothat outlined in FIG. 2 were observed.

FIG. 4. Nucleotide sequence alignment of representative angiogenin genesfrom different organisms (SEQ ID NOS: 4-12).

FIG. 5. Amino acid sequence alignment of representative angiogenin genesfrom different organisms (SEQ ID NOS: 13-21).

FIG. 6. Nucleotide sequence of the Bos taurus angiogenin, ribonuclease,RNase A family, 5 (ANG), minus its signal peptide sequence, modified formonocot plant codon bias (SEQ ID NO: 22).

FIG. 7. Nucleotide sequence of the Bos taurus angiogenin, ribonuclease,RNase A family, 5 (ANG), minus its signal peptide sequence, modified fordicot plant codon bias (SEQ ID NO: 23).

FIG. 8. Nucleotide sequence alignment, indicating 80.7% similarity,between ANG modified for monocot and dicot plant codon bias. No changesin amino acid sequence to that outlined in FIG. 2 were observed.

FIG. 9. Nucleotide sequence of Arabidopsis oleosin_ANG fusion gene (SEQID NO: 24). The Arabidopsis olesin gene is indicated in plain UPPERCASE.The thrombin protease recognition site is highlighted in

followed by the ANG gene in underlined UPPERCASE font. The start andstop codons are highlighted in

.

FIG. 10. Amino acid sequence of the Arabidopsis oleosin_ANG fusionprotein (SEQ ID NO: 25). The Arabidopsis olesin protein is indicated inplain UPPERCASE. The thrombin protease recognition site is highlightedin

italics followed by the ANG protein in underlined UPPERCASE font.

FIG. 11. Nucleotide sequence of an expression cassette containing theANG gene with an ER signal retention peptide regulated by the AtRbcSlight regulated promoter and nopaline synthase (nos) terminator foraccumulation in dicot plant tissue (SEQ ID NO: 26). The expressioncassette contains the dicot optimised ANG gene sequence outlined in FIG.7. The AtRbcS promoter is indicated in UPPERCASE italics, the ANG geneis in plain UPPERCASE with the ER signal retention peptide UNDERLINEDand the start and stop codon highlighted in

. The nos terminator is presented in lowercase.

FIG. 12. Vector map of sequence outlined in FIG. 11 containing the ANGgene with an ER signal retention peptide regulated by the AtRbcS lightregulated promoter and nos terminator for transfection and accumulationin dicot plant tissue.

FIG. 13. Vector map of a control expression cassette designed to expressthe fluorescent reporter (turboGFP) under control of the constitutiveCaMV35s promoter from the plant Cauliflower Mosaic virus (CaMV) forconfirmation of expression in dicot plant tissue.

FIG. 14. Nucleotide sequence of an expression cassette containing theANG gene with an ER signal retention peptide regulated by the TaRbcSlight regulated promoter and nopaline synthase (nos) terminator foraccumulation in monocot plant tissue (SEQ ID NO: 27). The expressioncassette contains the monocot optimised ANG gene sequence outlined inFIG. 6. The TaRbcS promoter is indicated in UPPERCASE italics, the ANGgene is in plain UPPERCASE with the ER signal retention peptideUNDERLINED and the start and stop codon highlighted in

. The nos terminator is presented in lowercase.

FIG. 15. Vector map of sequence outlined in FIG. 14 containing the ANGgene with an ER signal retention peptide regulated by the TaRbcS lightregulated promoter and nos terminator for accumulation in monocot planttissue.

FIG. 16. Vector map of a control expression cassette designed to expressthe fluorescent reporter (dsRED) under control of the constitutiveubiquitin promoter from Zea mays (ZmUbi) for confirmation of expressionin monocot plant tissue.

FIG. 17. Nucleotide sequence of an expression cassette containing theANG gene with an ER signal retention peptide regulated by the AtRbcSlight regulated promoter and CaMV35S terminator for transformation andaccumulation in dicot plant tissue (SEQ ID NO: 28). The expressioncassette contains the ANG gene sequence outlined in FIG. 3. The AtRbcSpromoter is indicated in UPPERCASE ITALICS, the ANG gene is in plainUPPERCASE with the ER signal retention peptide UNDERLINED and the startand stop codon highlighted in

. The CaMV35S terminator is presented in lowercase.

FIG. 18. Vector map of sequence outlined in FIG. 11 containing the ANGgene with an ER signal retention peptide regulated by the AtRbcS lightregulated promoter and nos terminator for transformation andaccumulation in monocot plant tissue. The base vector sequence containsthe necessary elements for Agrobacterium-mediated transformation andregeneration under appropriate selection.

FIG. 19. Representative nucleotide sequence of an expression cassettecontaining the ANG gene with an ER signal retention peptide regulated bythe TaRbcS light regulated promoter and terminator for accumulation inmonocot plant tissue (SEQ ID NO: 29). The expression cassette containsthe ANG gene sequence outlined in FIG. 6. The TaRbcS promoter isindicated in UPPERCASE italics, the ANG gene is in plain UPPERCASE withthe ER signal retention peptide UNDERLINED and the start and stop codonhighlighted in

. The TaRbcS terminator is presented in lowercase.

FIG. 20. Vector map of sequence outlined in FIG. 14 containing the ANGgene with an ER signal retention peptide regulated by the TaRbcS lightregulated promoter and nos terminator for transformation andaccumulation in dicot plant tissue. The base vector sequence containsthe necessary elements for regeneration under appropriate selection.

FIG. 21. Representative nucleotide sequence of an expression cassettecontaining the ANG gene with an ER signal retention peptide regulated bythe LpRbcS light regulated promoter and LpFT4 terminator foraccumulation in monocot plant tissue (SEQ ID NO: 30). The expressioncassette contains the ANG gene sequence outlined in FIG. 6. The LpRbcSpromoter is indicated in UPPERCASE italics, the ANG gene is in plainUPPERCASE with the ER signal retention peptide UNDERLINED and the startand stop codon highlighted in

. The LpFT4 terminator is presented in lowercase.

FIG. 22. Representative nucleotide sequence of an expression cassettecontaining the ANG gene with an ER signal retention peptide regulated bythe Brassica napus napin gene seed specific promoter and CaMV35Sterminator for accumulation in dicot seeds (SEQ ID NO: 31). Theexpression cassette contains the ANG gene sequence outlined in FIG. 7.The napin gene promoter is indicated in UPPERCASE italics, the ANG geneis in plain UPPERCASE with the ER signal retention peptide UNDERLINEDand the start and stop codons highlighted in

. The CaMV35S terminator is presented in lowercase.

FIG. 23. Nucleotide sequence of an expression cassette containing theANG gene with an ER signal retention peptide regulated by the Brassicanapus napin gene seed specific promoter and nos terminator foraccumulation in dicot seeds (SEQ ID NO: 32). The expression cassettecontains the ANG gene sequence outlined in FIG. 7. The Bn_napin genepromoter is indicated in UPPERCASE italics, the ANG gene is in plainUPPERCASE with the ER signal retention peptide UNDERLINED and the startand stop codons highlighted in

. The nos terminator is presented in lowercase.

FIG. 24. Vector map of sequence outlined in FIG. 23 containing the ANGgene with an ER signal retention peptide regulated by the Brassica napusnapin promoter and nos terminator for transformation and accumulation indicot seed tissue. The base vector sequence contains the necessaryelements for Agrobacterium-mediated transformation and regenerationunder appropriate selection.

FIG. 25. Representative nucleotide sequence of an expression cassettecontaining the ANG gene with an ER signal retention peptide regulated bythe Zea mays zein 4 gene seed specific promoter and CaMV35S terminatorfor accumulation in monocot seeds (SEQ ID NO: 33). The expressioncassette contains the ANG gene sequence outlined in FIG. 6. The Zm zeinpromoter is indicated in UPPERCASE italics, the ANG gene is in plainUPPERCASE with the ER signal retention peptide UNDERLINED and the startand stop codons highlighted in

. The CaMV35S terminator is presented in lowercase.

FIG. 26. Nucleotide sequence of an expression cassette containing theANG gene with an ER signal retention peptide regulated by the Zea mayszein 4 gene seed specific promoter and nos terminator for accumulationin monocot seeds (SEQ ID NO: 34). The expression cassette contains theANG gene sequence outlined in FIG. 6. The Zm zein 4 gene promoter isindicated in UPPERCASE italics, the ANG gene is in plain UPPERCASE withthe ER signal retention peptide UNDERLINED and the start and stop codonshighlighted in

. The nos terminator is presented in lowercase.

FIG. 27. Vector map of sequence outlined in FIG. 26 containing the ANGgene with an ER signal retention peptide regulated by the Zea mays zein4 promoter and nos terminator for transformation and accumulation inmonocot seed tissue. The base vector sequence contains the necessaryelements for Agrobacterium-mediated transformation and regenerationunder appropriate selection.

FIG. 28. Nucleotide sequence of an expression cassette containing theANG gene with an ER signal retention peptide regulated by the Oryzasativa PR602 gene seed specific promoter and nos terminator foraccumulation in monocot seeds (SEQ ID NO: 35). The expression cassettecontains the ANG gene sequence outlined in FIG. 6. The PR602 genepromoter is indicated in UPPERCASE italics, the ANG gene is in plainUPPERCASE with the ER signal retention peptide UNDERLINED and the startand stop codons highlighted in

. The nos terminator is presented in lowercase.

FIG. 29. Vector map of sequence outlined in FIG. 28 containing the ANGgene with an ER signal retention peptide regulated by the Oryza sativaPR602 promoter and nos terminator for transformation and accumulation inmonocot seed tissue. The base vector sequence contains the necessaryelements for Agrobacterium-mediated transformation and regenerationunder appropriate selection.

FIG. 30. Nucleotide sequence of an expression cassette containing theANG gene with an ER signal retention peptide regulated by the Triticumaestivum glutelin gene seed specific promoter and nos terminator foraccumulation in monocot seeds (SEQ ID NO: 36). The expression cassettecontains the ANG gene sequence outlined in FIG. 6. The glutelin genepromoter is indicated in UPPERCASE italics, the ANG gene is in plainUPPERCASE with the ER signal retention peptide UNDERLINED and the startand stop codons highlighted in

. The nos terminator is presented in lowercase.

FIG. 31. Vector map of sequence outlined in FIG. 30 containing the ANGgene with an ER signal retention peptide regulated by the Triticumaestivum glutelin promoter and nos terminator for transformation andaccumulation in monocot seed tissue. The base vector sequence containsthe necessary elements for Agrobacterium-mediated transformation andregeneration under appropriate selection.

FIG. 32. Representative nucleotide sequence of an expression cassettecontaining the ANG gene with the tobacco calreticulin apoplast signalpeptide regulated by the constitutive CaMV35S promoter and terminatorfor guttation secretion in plants (SEQ ID NO: 37). The expressioncassette contains the ANG gene sequence outlined in FIG. 7. The CaMV35Spromoter is indicated in UPPERCASE italics, the ANG gene is in plainUPPERCASE with the apoplast signal peptide UNDERLINED and the start andstop codons highlighted in

. The CaMV35S terminator is presented in lowercase.

FIG. 33. Representative nucleotide sequence of an expression cassettecontaining the ANG gene with the tobacco calreticulin apoplast signalpeptide regulated by the Arabidopsis phosphate transporter (AtPHT1) generoot-specific promoter and CaMV35S terminator for secretion in dicotroots (SEQ ID NO: 38). The expression cassette contains the ANG genesequence outlined in FIG. 7. The AtPHT1 promoter is indicated inUPPERCASE italics, the ANG gene is in plain UPPERCASE with the apoplastsignal peptide UNDERLINED and the start and stop codons highlighted in

. The CaMV35S terminator is presented in lowercase.

FIG. 34. Representative nucleotide sequence of an expression cassettecontaining the ANG gene with the tobacco calreticulin apoplast signalpeptide regulated by the Hordeum vulgare phosphate transporter (HvPHT1)gene root-specific promoter and CaMV35S terminator for secretion inmonocot roots (SEQ ID NO: 39). The expression cassette contains the ANGgene sequence outlined in FIG. 6. The HvPHT1 promoter is indicated inUPPERCASE italics, the ANG gene is in plain UPPERCASE with the apoplastsignal peptide UNDERLINED and the start and stop codons highlighted in

. The CaMV35S terminator is presented in lowercase.

FIG. 35. Representative nucleotide sequence of an expression cassettecontaining an oleosin_ANG gene fusion regulated by the Arabidopsisoleosin gene promoter and CaMV35S terminator for targeting to theoilbody in dicots (SEQ ID NO: 40). The expression cassette contains theANG gene sequence outlined in FIG. 7. The Arabidopsis oleosin genepromoter is indicated in UPPERCASE italics. The Arabidopsis olesin geneis indicated in plain UPPERCASE and the ANG gene in underlined UPPERCASEwith the thrombin protease recognition site highlighted in

and the start and stop codons highlighted in

. The CaMV35S terminator is presented in lowercase.

FIG. 36. Representative nucleotide sequence of an expression cassettecontaining the tobacco 16sRNA operon (Prrn) promoter and terminatorregulatory sequences (Zoubenko, et al., 1994) to express the angiogeningene in chloroplasts (SEQ ID NO: 41). The 16sRNA operon (Prrn) promoteris indicated in UPPERCASE italics, the ANG gene is in plain UPPERCASEand the start and stop codons highlighted in

. The 16sRNA operon (Prrn) terminator is presented in lowercase.

FIG. 37. A. Mesophyll-derived protoplasts of Nicotiana tabacum recoveredfrom in-vitro grown leaves approximately 4-6 weeks old; 0 days posttransfection; B. Assessment of protoplasts vigour, with dead cellsindicated by dark staining, showing greater than 95 percent viabilityusing Evan's Blue Stain; pre-transfection; C. Assessment of protoplastvigour, with dead cells indicated by dark staining, showing greater than95 percent viability using Evan's Blue Stain; 36 hourspost-transfection.

FIG. 38. Assessment of transient expression 36 hours post transfectionwith plasmid DNA containing the turboGFP gene encoding the greenfluorescent protein. Protoplasts visualised under A. bright field and B.fluorescent light. The green fluorescent protein is observed as a brightspot under fluorescent light.

FIG. 39. Electrophoresis of Reverse-transcriptase PCR samples andcontrols. Lane 1: NO-RT control reaction performed with ANG (F and R)primers on tobacco mesophyll protoplasts transfected with 0957286CaMV35S-p_turboGFP_nos-t. Lane 2: cDNA from tobacco mesophyllprotoplasts transfected with 0957286 CaMV35S-p_turboGFP_nos-t amplifiedwith ANG (F and R) primers. Lane3: NO-RT control reaction performed withANG (F and R) primers on tobacco mesophyll protoplasts transfected with1031308 AtRbcS-p_ANG_nos-t. Lane 4: cDNA from tobacco mesophyllprotoplasts transfected with 1031308 AtRbcS-p_ANG_nos-t amplified withANG (F and R) primers. Lane 5: Negative control reaction performedwithout template (ANG F and R primers). Lane 6: Positive controlreaction performed with plasmid template (ANG F and R primers). Lane 7:1 kb plus DNA Ladder (Invitrogen) Lane 8: NO-RT control reactionperformed with Actin (F and R) primers on tobacco mesophyll protoplaststransfected with 0957286 CaMV35S-p_turboGFP_nos-t. Lane 9: cDNA fromtobacco mesophyll protoplasts transfected with 0957286CaMV35S-p_turboGFP_nos-t amplified with Actin (F and R) primers. Lane10: NO-RT control reaction performed with Actin (F and R) primers ontobacco mesophyll protoplasts transfected with 1031308AtRbcS-p_ANG_nos-t. Lane 11: cDNA from tobacco mesophyll protoplaststransfected with 1031308 AtRbcS-p_ANG_nos-t amplified with Actin (F andR) primers. Lane 12: Negative control reaction performed withouttemplate (Actin F and R primers).

FIG. 40. A. Mesophyll-derived protoplasts recovered from mature leavesof T. aestium; 0 days post transfection; B. Assessment of protoplastvigour, with dead cells indicated by dark staining, showing greater than95 percent viability using Evan's Blue Stain; pre-transfection; C.Assessment of protoplast vigour, with dead cells indicated by darkstaining, showing greater than 81 percent viability using Evan's BlueStain; 24 hours post-transfection.

FIG. 41. Assessment of transient expression 36 hours post transfectionwith plasmid DNA containing the dsRED gene encoding the dsRED protein.Protoplasts visualised under A. bright field and B. fluorescent light.The dsRED protein is observed as a bright spot under fluorescent light.

FIG. 42. Electrophoresis of Reverse-transcriptase PCR samples andcontrols. Lane 1: NO-RT control reaction performed with ANG_F andpolyT_R primers on wheat mesophyll protoplasts. Lane 2: cDNA generatedby reverse transcription with oligo-dT from total RNA of wheat mesophyllprotoplasts amplified with ANG_F and polyT_R primers. Lane3: NO-RTcontrol reaction performed with ANG_F and polyT_R primers on wheatmesophyll protoplasts transfected with 1031312_TaRbcS-p_ANG_nos-t. Lane4: cDNA from wheat mesophyll protoplasts transfected with1031312_TaRbcS-p_ANG_nos-t amplified with ANG_F and polyT_R primers.Lane 5: Negative control reaction performed without template (ANG_F andpolyT_R primers). Lane 6: 1 kb plus DNA Ladder (Invitrogen). Lane 7:NO-RT control reaction performed with Actin_F and polyT_R primers onwheat mesophyll protoplasts. Lane 8: cDNA from wheat mesophyllprotoplasts amplified with Actin_F and polyT_R primers. Lane 9: NO-RTcontrol reaction performed with Actin_F and polyT_R primers on tobaccomesophyll protoplasts transfected with 1031308 AtRbcS-p_ANG_nos-t. Lane10: cDNA from wheat mesophyll protoplasts transfected with1031312_TaRbcS-p_ANG_nos-t amplified with Actin_F and polyT_R primers.Lane 11: Negative control reaction performed without template (Actin_Fand polyT_R primers).

FIG. 43. Agrobacterium-mediated transformation of Canola (Brassicanapus): A. seed imbibed on filter paper support; B. synchronousgermination of seed; C. pre-processing of germinated shoots; D,processing of cotyledons for use as explants; E. regeneration of shootsfollowing cocultivation with Agrobacterium; and F. mature plant inglasshouse.

FIG. 44. Preparation of embryogenic callus and biolistic transformationof perennial ryegrass: A. tillers of flowering glasshouse-grown plantsprior to surface-sterilisation; B. an immature inflorescence isolatedfor culture in vitro; C. embryogenic callus after culturing of immatureinflorescence tissue in vitro for 4-6 weeks; D-E. isolation of 3-5 mmexplants of friable embryogenic callus prior to particle bombardment; F.biolistic bombardment of callus with gold particles coated with atransformation construct; G-H. an antibiotic-resistant shoot onselective medium; I. antibiotic-resistant shoots in vessels ofroot-inducing medium; J. putative transgenic plantlets in soil.

FIG. 45. Agrobacterium-mediated transformation of bread wheat: A. donorplants ready for harvest; B&C. harvested material for use as source ofembryo explants; D. callus material; E. pre-regeneration material ontissue culture medium; F. callus material illustrating reporter geneexpression; G. regenerating shoots from callus; H. rooting shoots onselection media; and I. rooted plant in soil.

FIG. 46. Agrobacterium-mediated transformation of white clover: A.isolation of cotyledonary explants from a mature seed; B. selection ofantibiotic-resistant shoots on regeneration medium, C.antibiotic-resistant shoots in vessels of root-inducing medium and D. aputative transgenic plantlet in soil.

FIG. 47. Map of transformation vector containing nos_nptII_nosselectable marker cassette and the AtRbcS_ANG_CamV35S (FIG. 17)expression cassette used in Agrobacterium mediated transformation ofwhite clover.

FIG. 48. RT-PCR of positive and negative control (lanes 5 and 6) andputative transgenic angiogenin white clover plants (lanes 1 to 4).Primers used were specific to the angiogenin gene.

FIG. 49. 2DE gel protein analysis of non-transgenic control andtransgenic white clover plants. The three circles represent the ribulosebisphosphate carboxylase small subunit. The angiogenin protein isrepresented by the square.

FIG. 50. 2DE gel protein sequence analysis (SEQ ID NO: 42). Sixty sevenpercent sequence coverage (indicated in bold and underlined) wasobtained of the protein extracted from the gel.

FIG. 51. Electrophoresis of PCR samples and controls. Lane 1: 1 kb plusDNA Ladder (Invitrogen) Lane 2 and 3: PCR of DNA from Arabidopsistransgenic line 1, transformed with pPFG000023 AtRbcS-ANG_nos-t,amplified with ANG (F and R) primers. Lane 4 and 5: PCR of DNA fromArabidopsis transgenic line 2, transformed with pPFG000023AtRbcS-ANG_nos-t, amplified with ANG (F and R) primers. Lane 6 and 7:PCR of DNA from wild-type untransformed Arabidopsis amplified with ANG(F and R) primers. Lane 8 and 9: Positive control reaction performedwith pPFG000023 plasmid template (ANG F and R primers).

DETAILED DESCRIPTION OF THE EMBODIMENTS

In a first aspect, the present invention provides a plant cell, plantcallus, plant, seed or other plant part including an angiogenin gene ora functionally active fragment or variant thereof and/or an angiogeninpolypeptide. Preferably, said plant cell, plant callus, plant, seed orother plant part is produced by a method as described herein.

In a preferred aspect, the angiogenin gene or functionally activefragment or variant thereof may be co-expressed with a modular ormediator of angiogenin activity.

By ‘plant cell’ is meant any self-propagating cell bounded by asemi-permeable membrane and containing a plastid. Such a cell alsorequires a cell wall if further propagation is desired. Plant cell, asused herein includes, without limitation, algae, cyanobacteria, seedssuspension cultures, embryos, meristematic regions, callus tissue,leaves, roots, shoots, gametophytes, sporophytes, pollen andmicrospores.

In a second aspect, the present invention provides methods of using theplant cells, plant calli, plants, seeds or other plant parts includingan angiogenin as a composition such as a feed stock, food supplement orveterinary product for animals or a food, food supplement, nutraceuticalor pharmaceutical suitable for human consumption. For example, the valueadded plant material, including the angiogenin protein, may be used asan enhanced feedstock for a variety of applications.

Accordingly, the present invention provides a method of using a plantcell, plant callus, plant, seed or other plant part including anangiogenin as feed stock for animals or as a composition suitable forhuman consumption, said method comprising producing the angiogenin inthe plant cell, plant callus, plant, seed or other plant part andpreparing it in a form suitable for use as a feed stock for animals or acomposition suitable for human consumption.

Animals to which the invention may be applied include pigs, chickens(broilers and layers), beef, dairy, goats, sheep are livestock, that canbenefit from abundant sources of angiogenin provided by plants, as wouldcompanion animals and performance animals eg horses, dogs.

It may be desirable to administer plant derived angiogenin encapsulatedor otherwise protected to passage the rumen or stomach more effectively.Less digestible tissues such as seed coat and roots (as opposed to fruitand leaves) may extend gut passage and digestive tract protein releasefor intestinal binding and uptake.

Co-administration with other supplements and treatments, eg growthhormone such as bovine somatotrophin, antibiotics, nutrient supplementsfor animals, is also contemplated.

In a third aspect, the present invention provides a plant-producedangiogenin. Preferably said angiogenin is produced by a method asdescribed herein.

In a further aspect, the present invention provides a feedstock, foodsupplement or veterinary product including a plant-produced angiogenin.Preferably said angiogenin is produced by a method as described herein.

In a further aspect, the present invention provides a food, beverage,food supplement, nutraceutical or pharmaceutical including aplant-produced angiogenin. Preferably said angiogenin is produced by amethod as described herein.

In a further aspect, the present invention provides a method ofproducing a transformed plant cell expressing an angiogenin gene, saidmethod comprising

-   -   providing a gene encoding angiogenin or a functionally active        fragment or variant thereof, and a plant cell;    -   introducing the angiogenin gene into the plant cell to produce a        transformed plant cell; and    -   culturing the transformed plant cell to produce a transformed        plant cell expressing the angiogenin gene.

By a ‘transformed plant cell’ is meant a plant cell which has undergonetransformation.

By ‘transformation’ is meant the transfer of nucleic acid into a plantcell.

By a ‘gene encoding angiogenin” or ‘angiogenin gene’ is meant a nucleicacid encoding a polypeptide having one or more of the biologicalproperties of angiogenin. The gene encoding angiogenin may be atransgene. The gene encoding angiogenin may include an angiogenin codingsequence optionally operatively linked to a sequence encoding one ormore of a promoter, signal peptide, terminator, and mediator ormodulator of angiogenin activity.

By a ‘transgene’ is meant a nucleic acid suitable for transforming aplant cell.

By a ‘functionally active’ fragment or variant of an angiogenin gene ismeant that the fragment or variant (such as an analogue, derivative ormutant) encodes a polypeptide having one or more of the biologicalproperties of angiogenin. Such variants include naturally occurringallelic variants and non-naturally occurring variants. Additions,deletions, substitutions and derivatizations of one or more of thenucleotides are contemplated so long as the modifications do not resultin loss of functional activity of the fragment or variant.

Preferably the functionally active fragment or variant has at leastapproximately 80% identity to the relevant part of the specifiedsequence to which the fragment or variant corresponds, more preferablyat least approximately 90% identity, even more preferably at leastapproximately 95% identity, most preferably at least approximately 98%identity.

Preferably the fragment has a size of at least 20 nucleotides, morepreferably at least 50 nucleotides, more preferably at least 100nucleotides, more preferably at least 200 nucleotides, more preferablyat least 300 nucleotides.

Such functionally active variants and fragments include, for example,those having conservative nucleic acid changes, those having codon usageadapted for plants, and those in which the signal peptide is removed andoptionally replaced by another signal peptide.

By ‘conservative nucleic acid changes’ is meant nucleic acidsubstitutions that result in conservation of the amino acid in theencoded protein, due to the degeneracy of the genetic code. Suchfunctionally active variants and fragments also include, for example,those having nucleic acid changes which result in conservative aminoacid substitutions of one or more residues in the corresponding aminoacid sequence.

By ‘conservative amino acid substitutions’ is meant the substitution ofan amino acid by another one of the same class, the classes being asfollows:

-   -   Nonpolar: Ala, Val, Leu, Ile, Pro, Met Phe, Trp    -   Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln    -   Acidic: Asp, Glu    -   Basic: Lys, Arg, His

Other conservative amino acid substitutions may also be made as follows:

-   -   Aromatic: Phe, Tyr, His    -   Proton Donor Asn, Gln, Lys, Arg, His, Trp    -   Proton Acceptor Glu, Asp, Thr, Ser, Tyr, Asn, Gln

Particularly preferred fragments and variants include one or moreconserved binding domains such as sequences encoding a catalytic core ora cell binding site. Examples of such domains are shown in FIG. 2 andpreferably include the sequence Arg, Asn, Gly, Gln, Pro, Tyr, Arg, Gly,Asp (SEQ ID NO: 43).

Particularly preferred fragments and variants include a catalytic core.By a “catalytic core” is meant an internal region of the polypeptideexcluding signal peptide and N- and C-terminal variable regionsincluding catalytic amino acids. Examples of catalytic amino acids areshown in FIG. 2.

Two distinct regions of angiogenin are required for its angiogenicactivity including a catalytic site containing His-13, Lys-41, andHis-115 that is capable of cleaving RNA and a noncatalytic, cell bindingsite encompassing minimally residues 60-68. RNase activity and receptorbinding capacity, while required, are not sufficient for angiogenicactivity: endocytosis and nuclear translocation are required as well.

Catalytic residues in angiogenin include His-13, Lys-40, Gln-12 andThr-44, for example. These residues may be conserved to retain RNaseand/or cellular activity.

Activity may be increased or decreased by changing key amino acids at ornear the active site with improved activity substituting Asp-116 to Hisbeing an example (Acharva, Shapiro et al). Arg-5 and Arg-33 may also beimportant for activity.

Cellular uptake of angiogenin in proliferating endothelial cells ismediated by domains and is not dependent upon RNase activity asenzymatically inactive mutants can be internalized. K41Q and H13Amutants for example are enzymatically inactive but are translocated.Improved versions of angiogenin more readily internalised by cells andmore potent are within the scope of the present invention, and suchvariants can be tested for by conducting in vitro uptake and activitytests on epithelial and muscle cells in culture.

Particularly preferred fragments and variants include those lacking asignal peptide. By a “signal peptide” is meant an N-terminal signalsequence. An example of a signal peptide is shown in FIG. 2 and includesthe sequence Met, Val, Met, Val, Leu, Ser, Pro, Leu, Phe, Leu, Val, Phe,Ile, Leu, Gly, Leu, Gly, Leu Thr, Pro, Val, Ala, Pro, Ala (SEQ ID NO:44).

Particularly preferred fragments and variants have codon usage adaptedfor plants, including the start of translation for monocots and dicots.Thus, the fragment or variant encodes a polypeptide having one or moreof the biological properties of angiogenin, but one or more codons,particularly in the third position, may be changed so that the gene ismore readily expressed in plants compared with the corresponding animalgene. Changes to one or more of the nucleotides are contemplated so longas the modifications do not result in loss of functional activity of thefragment or variant. Preferably the fragment or variant has at leastapproximately 60% identity to the relevant part of the original animalsequence to which the modified gene corresponds, more preferably atleast approximately 80% identity, even more preferably at leastapproximately 95% identity, most preferably at least approximately 98%identity. Particularly preferred fragments and variants have crypticsplice sites and/or RNA destabilizing sequence elements inactivated orremoved.

It may also be desirable to remove A+T—rich sequences that may causemRNA instability. This may increase mRNA stability or aberrant splicingand improve efficiency of transcription in plant cell nuclei. This mayalso eliminate a potential premature poly(A).

Preferably, the angiogenin gene is isolated from or corresponds to anangiogenin gene from an animal, more preferably from a cow, human,gorilla, chimp, monkey, horse, pig, rat, mouse, fish or chicken, evenmore preferably from Bos taurus (cow).

In a particularly preferred embodiment the angiogenin gene encodes apolypeptide comprising the sequence shown in FIG. 2.

In another particularly preferred embodiment, the angiogenin genecomprises a sequence selected from the group consisting of the sequencesshown in FIG. 4; and functionally active fragments and variants thereof.

To reduce the possibility of aberrant developmental phenotypes theangiogenin gene may be modified to alter its targeting signal sequenceto direct the angiogenin gene to a target sub-cellular component orplant tissue, such as ER, apoplast, peroxisome or vacuole.

More particularly, a chimeric sequence may be created, whereby thesignal peptide of the angiogenin gene may be removed and optionallyreplaced by another signal peptide, for example a plant signal peptide,said plant signal peptide optionally driving angiogenin accumulation toa selected sub-cellular component or plant tissue.

Accordingly, in a still further aspect, the present invention provides achimeric sequence comprising an angiogenin gene, or a functionallyactive fragment or variant thereof, and a plant signal peptide.

In a preferred embodiment, the plant signal peptide may be from orcorrespond to a signal peptide from an ER-derived protein, such as aprotein containing a C-terminus 4-amino-acid retention sequence, KDEL(lys-asp-glu-leu).

The angiogenin gene may be introduced into the plant cell by anysuitable technique. Techniques for incorporating the angiogenin geneinto plant cells (for example by transduction, transfection,transformation or gene targeting) are well known to those skilled in theart. Such techniques include Agrobacterium-mediated introduction,Rhizobium-mediated introduction, electroporation to tissues, cells andprotoplasts, protoplast fusion, injection into reproductive organs,injection into immature embryos and high velocity projectileintroduction to cells, tissues, calli, immature and mature embryos,biolistic transformation, Whiskers transformation, and combinationsthereof.

The choice of technique will depend largely on the type of plant cell tobe transformed, and may be readily determined by an appropriatelyskilled person.

The present invention may be applied to a variety of plants, includingmonocotyledons [such as grasses (e.g. forage grasses including perennialryegrass, tall fescue, Italian ryegrass, brachiaria, paspalum), sorghum,sugarcane, corn, oat, wheat, rice and barley)], dicotyledons [such asforage legumes (e.g. white clover, red clover, subterranean clover,alfalfa), soybean, lupin, peas, lentils, chickpeas, canola, vegetablebrassicas, lettuce, spinach, fruiting plants (e.g. bananas, citrus,strawberries, apples), oil palm, linseed, cottonseed, safflower,tobacco] and gymnosperms.

In a further aspect the present invention provides a method of producingan angiogenin in a plant, said method comprising

-   -   providing a gene encoding angiogenin or a functionally active        fragment or variant thereof, and a plant cell;    -   introducing the angiogenin gene into the plant cell to produce a        transformed plant cell;    -   culturing the transformed plant cell to produce a transformed        plant cell expressing the angiogenin gene; and    -   isolating the angiogenin produced by the plant cell.

The angiogenin may be isolated by techniques known to those skilled inthe art. For example, cation exchange purification (or enrichment), orsize selection may be used.

The term “isolated” means that the angiogenin is removed from itsoriginal environment, and preferably separated from some or all of thecoexisting materials in the transformation system. Preferably, theangiogenin is at least approximately 90% pure, more preferably at leastapproximately 95% pure, even more preferably at least approximately 98%pure.

In a further aspect, the present invention provides a method ofproducing transformed plant calli, plants, seeds or other plant partsincluding angiogenin, said method comprising

-   -   providing a gene encoding angiogenin or a functionally active        fragment or variant thereof, and a plant cell;    -   introducing the angiogenin gene into the plant cell to produce a        transformed plant cell;    -   culturing the transformed plant cell to produce transformed        plant calli, plants, seeds or other plant parts including        angiogenin.

Cells incorporating the angiogenin gene may be selected, as describedbelow, and then cultured in an appropriate medium to regeneratetransformed plant calli, plants, seeds or other plant parts, usingtechniques well known in the art. The culture conditions, such astemperature, pH and the like, will be apparent to the person skilled inthe art. The resulting plants may be reproduced, either sexually orasexually, using methods well known in the art, to produce successivegenerations of transformed plants.

In a further aspect, the method further includes isolating angiogeninfrom the transformed plant calli, plants, seeds or other plant parts.

The angiogenin may be isolated by techniques known to those skilled inthe art, for example by extraction. For example, angiogenin may beisolated from ultrafiltrate (Fedorova et al., 2002), includingprecipitation with ammonium sulfate, followed by cation exchangepurification, or using a placental ribonuclease inhibitor binding assay(Bond and Vallee, 1988). More purification may be required for humanapplications and processed food ingredients and construction.

In a still further aspect, the present invention provides methods ofenhancing expression, activity or isolation of angiogenin in plants. Theangiogenin gene may be modified to improve its function in animals,particularly mammals. Plant expression may be tailored for enhancedactive protein preparation, digestive uptake and biological activity inhumans and other animals. For example, the angiogenin gene may bemodified to improve a function selected from the group consisting ofcellular delivery, myogenic activity, RNase enzyme activity, rRNAtranscriptional activity and/or DNA binding activity, rRNA processingand/or splicing activity and receptor binding and/or endocytosis. Forexample, protease stability, heat stability and/or pH resistance may beimproved, which may in turn assist in processing and/or purification ofplant-produced angiogenin.

Post-harvest treatment and/or processing may also enhance heatstability, protease stability and/or, cellulase treatment compatibility.

The present invention also contemplates silage compatible expression inplants. Antimicrobial co-expression may be used to stabilize nativeprotein by protecting from or reducing bacterial and/or fungaldegradation. Examples include antimicrobial peptides made by bacteria(bacteriocins) or plants (eg thionines, plant defensins) or fungi (AFPand PAF from filamentous fungi) or animals (cathelicidins, defensins,lysozymes). Angiogenin may be complexed with RNase inhibitor to enhanceangiogenin expression when co-expressed to reduce toxicity in plants.

The present invention also contemplates co-expressing an angiogenin geneor functionally active fragment or variant thereof with a gene encodinga mediator or modulator of angiogenin activity.

By a ‘mediator or modulator of angiogenin activity’ is meant a moleculethat enhances or otherwise modifies expression, activity or isolation ofangiogenin in a plant cell, plant callus, plant, seed or other plantpart. For example, the mediator or modulator of angiogenin activity mayimprove protein accumulation, enhance protein action or activity, ormake isolation of the protein more effective. Other examples includeenhancement of post-harvest treatment, silage compatibility orprocessing, improvement of protease stability or heat stability andimprovement of treatment compatibility.

For example, the angiogenin gene may be co-expressed with a geneencoding one or more of antimicrobials, protease inhibitors, RNaseinhibitors, follistatin, and delayed plant organ senescence gene orgenes.

The present invention also contemplates artificial constructs orchimeric sequences comprising an angiogenin gene or functionally activefragment or variant thereof and a gene encoding a mediator or modulatorof angiogenin activity.

By a ‘chimeric sequence’ is meant a hybrid produced recombinantly byexpressing a fusion gene including two or more linked nucleic acidswhich originally encoded separate proteins, or functionally activefragments or variants thereof.

By a ‘fusion gene’ is meant that two or more nucleic acids are linked insuch a way as to permit expression of the fusion protein, preferably asa translational fusion. This typically involves removing the stop codonfrom a nucleic acid sequence coding for a first protein, then appendingthe nucleic acid sequence of a second protein in frame. The fusion geneis then expressed by a cell as a single protein. The protein may beengineered to include the full sequence of both original proteins, or afunctionally active fragment or variant of either or both.

The present invention also provides an angiogenin gene with codon usageadapted for plants, said angiogenin gene being capable of beingexpressed in a plant cell which has been transformed with said gene.

Preferably, the angiogenin gene is isolated from or corresponds to anangiogenin gene from an animal, more preferably Bos taurus (cow).

In a particularly preferred embodiment the angiogenin gene encodes apolypeptide comprising the sequence shown in FIG. 2.

In another particularly preferred embodiment, the angiogenin genecomprises a sequence selected from the group consisting of the sequencesshown in FIG. 4; and functionally active fragments and variants thereof.

By an ‘angiogenin gene with codon usage adapted for plants’ is meantthat the angiogenin gene encodes a polypeptide having one or more of thebiological properties of angiogenin, but that one or more codons,particularly in the third position, have been changed so that the geneis more readily expressed in plants compared with the correspondinganimal gene. Changes to one or more of the nucleotides are contemplatedso long as the modifications do not result in loss of functionalactivity of the fragment or variant. Preferably the angiogenin gene withcodon usage adapted for plants has at least approximately 60% identityto the relevant part of the original animal sequence to which themodified gene corresponds, more preferably at least approximately 80%identity, even more preferably at least approximately 95% identity, mostpreferably at least approximately 98% identity.

In a further aspect of the present invention, there is provided anartificial construct capable of enabling expression of an angiogeningene in a plant cell, said artificial construct including a promoter,operatively linked to an angiogenin gene, or a functionally activefragment or variant thereof.

By ‘artificial construct’ is meant a recombinant nucleic acid molecule.

By a ‘promoter’ is meant a nucleic acid sequence sufficient to directtranscription of an operatively linked nucleic acid sequence.

By ‘operatively linked’ is meant that the nucleic acid(s) and aregulatory sequence, such as a promoter, are linked in such a way as topermit expression of said nucleic acid under appropriate conditions, forexample when appropriate molecules such as transcriptional activatorproteins are bound to the regulatory sequence. Preferably an operativelylinked promoter is upstream of the associated nucleic acid.

By ‘upstream’ is meant in the 3′->5′ direction along the nucleic acid.

By ‘gene’ is meant a chain of nucleotides capable of carrying geneticinformation. The term generally refers to genes or functionally activefragments or variants thereof and or other sequences in the genome ofthe organism that influence its phenotype. The term ‘gene’ includes DNA(such as cDNA or genomic DNA) and RNA (such as mRNA or microRNA) that issingle- or double-stranded, optionally containing synthetic, non-naturalor altered nucleotide bases, synthetic nucleic acids and combinationsthereof.

In a preferred embodiment, the artificial construct according to thepresent invention may be a vector.

By a ‘vector’ is meant a genetic construct used to transfer geneticmaterial to a target cell.

The vector may be of any suitable type and may be viral or non-viral.The vector may be an expression vector. Such vectors includechromosomal, non-chromosomal and synthetic nucleic acid sequences, eg.derivatives of plant viruses; bacterial plasmids; derivatives of the Tiplasmid from Agrobacterium tumefaciens; derivatives of the Ri plasmidfrom Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes;bacterial artificial chromosomes; binary bacterial artificialchromosomes; vectors derived from combinations of plasmids and phageDNA. However, any other vector may be used as long as it is replicableor integrative or viable in the plant cell.

In a preferred embodiment of this aspect of the invention, theartificial construct may further include a terminator; said promoter,gene and terminator being operably linked.

The promoter, gene and terminator may be of any suitable type and may beendogenous to the target plant cell or may be exogenous, provided thatthey are functional in the target plant cell.

The promoter used in the constructs and methods of the present inventionmay be a constitutive, tissue specific or inducible promoter. Forexample, the promoter may be a constitutive cauliflower mosaic virus(CaMV35S) promoter for expression in many plant tissues, an inducible‘photosynthetic promoter’ (eg. ribulose 1,5-bisphosphate), capable ofmediating expression of a gene in photosynthetic tissue in plants underlight conditions, or a tissue specific promoter such as a seed specificpromoter, for example from a gene selected from the group consisting ofBrassica napus napin gene, Zea mays zein 4 gene, Oryza sativa PR602 geneand Triticum aestivum glutelin gene.

A variety of terminators which may be employed in the artificialconstructs of the present invention are also well known to those skilledin the art. The terminator may be from the same gene as the promotersequence or a different gene. Particularly suitable terminators arepolyadenylation signals, such as the CaMV35S polyA and other terminatorsfrom the nopaline synthase (nos) and the octopine synthase (ocs) genes.

The artificial construct, in addition to the promoter, the gene and theterminator, may include further elements necessary for expression of thegene, in different combinations, for example vector backbone, origin ofreplication (ori), multiple cloning sites, spacer sequences, enhancers,introns (such as the maize Ubiquitin Ubi intron), antibiotic resistancegenes and other selectable marker genes [such as the neomycinphosphotransferase (nptII) gene, the hygromycin phosphotransferase (hph)gene, the phosphinothricin acetyltransferase (bar or pat) gene], andreporter genes (such as beta-glucuronidase (GUS) gene (gusA)]. Theartificial construct may also contain a ribosome binding site fortranslation initiation. The artificial construct may also includeappropriate sequences for amplifying expression.

Those skilled in the art will appreciate that the various components ofthe artificial construct are operably linked, so as to result inexpression of the angiogenin gene. Techniques for operably linking thecomponents of the artificial construct of the present invention are wellknown to those skilled in the art. Such techniques include the use oflinkers, such as synthetic linkers, for example including one or morerestriction enzyme sites.

Preferably, the artificial construct is substantially purified orisolated. By ‘substantially purified’ is meant that the artificialconstruct is free of the genes, which, in the naturally-occurring genomeof the organism from which the nucleic acid or promoter of the inventionis derived, flank the nucleic acid or promoter. The term thereforeincludes, for example, an artificial construct which is incorporatedinto a vector; into an autonomously replicating plasmid or virus; orinto the genomic DNA of a prokaryote or eukaryote; or which exists as aseparate molecule (eg. a cDNA or a genomic or cDNA fragment produced byPCR or restriction endonuclease digestion) independent of othersequences. It also includes an artificial construct which is part of ahybrid gene encoding additional polypeptide sequence. Preferably, thesubstantially purified artificial construct is at least approximately90% pure, more preferably at least approximately 95% pure, even morepreferably at least approximately 98% pure.

The term “isolated” means that the material is removed from its originalenvironment (eg. the natural environment if it is naturally occurring).For example, a naturally occurring nucleic acid present in a livingplant is not isolated, but the same nucleic acid separated from some orall of the coexisting materials in the natural system, is isolated. Suchnucleic acids could be part of a vector and/or such nucleic acids couldbe part of a composition, and still be isolated in that such a vector orcomposition is not part of its natural environment.

As an alternative to use of a selectable marker gene to provide aphenotypic trait for selection of transformed host cells, the presenceof the artificial construct in transformed cells may be determined byother techniques well known in the art, such as PCR (polymerase chainreaction), Southern blot hybridisation analysis, histochemical assays(e.g. GUS assays), thin layer chromatography (TLC), northern and westernblot hybridisation analyses.

Applicant has surprisingly found that the methods of the presentinvention may result in enhanced yield of angiogenin in the transformedplant cell relative to yields of proteins typically produced intransgenic plant cells.

In a preferred embodiment the methods of the present invention provide ayield of between approximately 0.1% and 5%, more preferably betweenapproximately 5% and 10%, more preferably between approximately 10% and30%, of total soluble protein.

EXAMPLES Example 1 Cloning of the Bovine Angiogenin Gene

The Bos taurus (cow) angiogenin, ribonuclease, RNase A family, 5 (ANG),mRNA sequence is available from the National Centre for BiotechnologyInformation (NCBI), accession number AM_(—)0011078144. The predictedopen reading frame (ORF) contains 444 base pairs (bp) (FIG. 1) encodinga 148 amino acid (aa) (FIG. 2) sequence. Using the SignalP 3.0 server topredict the presence and location of signal peptide cleavage sites inamino acid sequences a 24 aa (72 bp) signal peptide sequence wasidentified (FIGS. 1 and 2).

The angiogenin protein sequence has been analysed by comparison to adatabase of known allergens, the Food Allergy and Resource ResearchProgram at the University of Nebraska allergen protein database (FARRPAllergen Online version 7.0). A BLASTp for every 80 amino acid peptidescontained within the protein was searched against the FAARP AllergenOnline database. None of the amino acid peptides contained 35% or higheridentity to any of the known allergens of the database, a standard oftenused as a threshold for allergenicity concern. A BLASTp for theangiogenin protein in its entirety was also searched against the FARRPAllergen Online dataset. The angiogenin protein did not contain eight ormore consecutive amino acids in common with any member of the database,a standard frequently used as a threshold for allergenicity concern.

Using the angiogenin NCBI sequence, primers were designed to amplify amodified ANG gene adapted for plant codon usage as defined by Murray etal. (1989) (FIG. 3). No changes in amino acid sequence to that outlinedin FIG. 2 were observed.

The Angiogenin Gene from Divergent Organisms

Using the bovine angiogenin gene as a query sequence a range ofdifferent sequences have been identified and are available from NCBI.Nucleotide and amino acid sequence alignments of angiogenin fromdivergent organisms have been produced (FIGS. 4 and 5).

Codon Optimisation of Angiogenin Genes for Expression in Plants

Different ANG nucleotide sequences to those outlined in FIGS. 1 and 3,optimised by alternate methods for codon bias of both monocot and dicotplants have been produced to enhance protein expression in plants (FIGS.6 and 7). Negative cis-acting sites which may negatively influenceexpression were eliminated wherever possible and GC content was adjustedto prolong mRNA half life. An alignment to indicate the difference insequence homology between the monocot and dicot optimised sequences ispresented in FIG. 8. The degree of sequence homology between the twosequences is 80.7%. The codon optimisation undertaken did not alter theamino acid sequence translation that is outlined in FIG. 2 (without thesignal peptide sequence).

Example 2 Production of Fusion Proteins for Greater Accumulation,Enhanced Action, or Improved Extraction, of Angiogenin

It is possible to create fusion proteins of angiogenin with mediators ormodulators of its activity to assist in the improvement of proteinaccumulation, enhancement of protein action, or for effective extractionof the protein.

Fusion Proteins for Enhancing the Action of Angiogenin

Yeast two-hybrid technology has identified potential ANG-interactingmolecules (Goa and Xu, 2008) such as alpha-actin 2 (ACTN-2) (Hu et al.,2005), regulatory proteins such as follistatin (FS) (Goa et al., 2007)and extracellular matrix proteins such as fibulin-1 (Zhang et al.,2008). It is hypothesised that through interacting with ACTN-2, ANG mayregulate the movement or the cytokinesis of the cells, follistatin mayact as a regulator on angiogenin's actions and interaction between ANGand fibulins may facilitate cell adhesion.

Follistatin is known to have a role in muscle growth and regulatesmuscle cell development through binding and blocking myostatin, a TGFfamily member and potent negative regulator of myoblast growth anddifferentiation. In partnership with RNase5, follistatin can actdirectly and synergistically as a positive regulator of muscle growthand differentiation. It has been demonstrated that RNase5 activation ofmuscle cell growth and differentiation in vitro is enhanced byfollistatin (patent PCT/AU2009/000603). Creation of a translationalfusion of these two genes, codon optimised for expression in plants, canbe used to enhance the ability of angiogenin to control muscledevelopment.

The activity of angiogenin may be blocked by ribonuclease inhibitors.Co-expression of angiogenin with ribonuclease inhibitor both codonoptimised for expression in plants, may be used to regulate theintracellular activity of angiogenin and improve expression by reducingtoxicity in plants.

Fusion Proteins for the Improved Extraction of Angiogenin

Oleosins provide an easy way of purifying proteins which have beenproduced recombinantly in plants. Oleosins are structural proteins foundin a unique seed-oil storage organelle know as the oilbody. It issuggested that a central hydrophobic domain within the oleosin gene ismost likely to play a role in localisation to the oil body. Therefore,through covalent fusions with oleosin a recombinant protein can bedirected to the oil bodies allowing easy extraction. Abenes et al.(1997) showed that an Arabidopsis oleosin-GUS fusion protein could beexpressed and targeted to oil bodies in at least five species ofoilseeds. Consequently, the angiogenin protein may be directed to theoil body by the creation of an oleosin_angiogenin fusion sequence (FIGS.9 and 10). Incorporating a protease recognition site between the twosequences allows the oleosin to be cleaved from the protein of interest.

Example 3 Identification of Promoter Sequences for Targeted Expressionof Angiogenin

Promoters with tissue-specificity are required to drive expression oftransgenes in crops to target accumulation in particular tissues/organsand to avoid unwanted expression elsewhere. Therefore highly expressingbut yet tightly controlled promoters are desirable.

Tissue Specific or Regulated Promoters

The choice of promoters affects transgene expression concentration, aswell as developmental, tissue and cell specificity. Examples ofdifferent promoters to drive transgene expression for differentobjectives are presented in Table 1.

TABLE 1 Examples of different promoters to drive transgene expression.Targeted expression Gene promoter Organism Reference ConstitutiveConstitutive Ubiquitin, Ubi Zea mays (maize) Christensen et al. (1992)CaMV35S² Cauliflower Kay et al. mosaic virus (1987) Polyubiquitin, RUBQ2Oryza sativa (rice) Liu et al. (2003) Actin 1, OsAct1 Oryza sativa(rice) McElroy et al. (1990) Tissue Specific Tuber and Sucrosesynthetase, Sus4 Solanum tuberosum Lin et al. stolon specific (potato)(2008) Cathepsin D inhibitor gene, Solanum tuberosum Herbers Cathinh(potato) et al. (1994) Root and shoot Helicase -like genes, helA,Pseudomonas plasmid Zhang of sugar beet helB and helC et al. (2004) Rootspecific Phosphate Transporter Arabidopsis thaliana Koyama AtPHT1 etal., (2005) Phosphate Transporter Hordeum vulgare Schunnman HvPHT1(barley) et al., (2004) Seed specific β-conglycinin, a soybean Glycinemax (soybean) Chen et al. seed storage protein (1988) 11S seed storageprotein Coffea Arabica Marraccini gene (coffee bean) et al. (1999) Napingene Brasica napus (canola) Lee et al. (1991) Glutelin A Oryza staiva(rice) Hashizume et al. (2008) Glutelin Triticum aestivum Lamacchia(wheat) et al. (2001) Zein gene, ZmZ4 Zea mays (maize) Penderson et al.(1982) Schernthaner et al. (1988) Endoperm Specific, Oryza staiva (rice)Li et al., OsPR602 (2008) Seed—Aluerone Maize regulatory gene B- Zeamays (maize) Selinger Peru et al. (1998) Fruit specific Fruit specificE8 Tomato Ramierez et al. (2007) Phloem Sucrose synthase, Suc2 Zea mays(maize) Yang and Russell (1990) Xylem phenylalanine Nicotianabenthamiana Keller and ammonialyase gene 2, (tobacco) Baumgartner PAL2(1991) 4-coumarate: coenzyme A Nicotiana benthamiana Hauffe et al.ligase. 4CL (tobacco) (1993) Xylem—lignified cinnamoyl coenzymeAEucalyptus gunnii Baghdady cells reductase (CCR) and (Eucalyptus) et al.(2006) cinnamyl alcohol dehydrogenase (CAD2) Inducible Cold, dehydrationCalcium dependent protein Oryza sativa (rice) Wan et al. and salt stresskinases, OsCPK6, (2007) responsive OsCPK13, OsCPK25 Dehydration stressearly responsive to Arabidopsis thaliana Tran et al. dehydration stress,ERD1 (2004) Stress responsive Rd29A Arabidopsis thaliana Yamaguchi-Shinozaki and Shinozaki (1993) Sucrose responsive ADP-glucose Ipomoeabatatas Kwak et al. pyrophosphorylase, IbAGP1 (sweet potato) (2005)ADP-glucose Lycopersicon esculentum Li et al. pyrophosphorylase, LeAgp(tomato) (2001) S1 14-3-3 protein family, 16R Solanum tuberosum Szopa etal. (potato) (2003) Ethylene responsive ethelyene responsive Gossypiumhirsutum Jin and Lui binding elements, GhERF4 (cotton) (2008) Coldresponsive wcs120 Triticum aestivum Ouellet (wheat) et al. (1998)Dessication StDS2 Solanum tuberosum Doczi et al. responsive in (potato)(2005) leaves, flowers and green fruit LeDS2 Lycopersicon esculentumDoczi et al. (2005) Oxidative stress Peptide methionine Arabidopsisthaliana Romero et al. induced by high sulfoxide reductase A, (2006)light and ozone PMRSA Wound Wun1, proteinase inhibitor Solanum tuberosumSiebertz II genes of potato (potato) et al. (1989) Starch ADP GlucoseArabidopsis thaliana Stark et al. Pyrophosphorylase, ADPGlc 1992 Lightregulated Ribulose-1,5-bisphosphate Triticum aestivum (wheat), Zeng, etal., carboxylase/oxygenase Arabidopsis thaliana, (1995), Small subunit,TaRbcS, and Lolium perenne Sasanuma, (2001) AtRbcS, and LpRbcSrespectively respectively Chlorophyll a/b Binding Lolium perenne(ryegrass) Protein, LpCAB

Representative examples of promoters for light regulated, seed and rootspecific linked to the angiogenin gene are presented in FIGS. 11-34.

Example 4 Identification of Signal Peptide Sequences for TargetedExpression of Angiogenin

Signal peptides are short (3-60 amino acids long) peptide chains thatdirect the transport of a protein to different subcellular compartmentssuch as the nucleus, mitochondrial matrix, endoplasmic reticulum (ER),chloroplast, apoplast, vacuole and peroxisome.

Most proteins that are transported to the ER have a sequence consistingof 5-10 hydrophobic amino acids on the N-terminus. The majority of theseproteins are then transported from the ER to the Golgi apparatus unlessthese proteins have a C-terminus 4-amino-acid retention sequence, KDEL(lys-asp-glu-leu), which holds them in the ER.

The nucleus and nucleolus can be targeted with either a nuclearlocalization signal (NLS) or a nucleolar localization signal(abbreviated NoLS or NOS), respectively. The signal peptide that directsto the mitochondrial matrix is usually called the mitochondrialtargeting signal (MTS). There are two types (N- and C-terminus)peroxisomal targeting signals (PTS). PTS1, consists of three amino acidsat the C-terminus while PTS2, is made of a 9-amino-acid sequence presenton the N-terminus of the protein.

Constructs Containing Tissue Specific or Regulated Promoters

Signal peptides are desirable to target accumulation of recombinantproteins for extraction from plant secretions or plant tissue. Examplesof different signal peptides to drive target protein accumulation indifferent sub-cellular compartments are presented in Table 2.

TABLE 2 Examples of different signal peptide sequences for targetedtransgene expression. Signal target Gene signal peptide OrganismReference ER H/KDEL (C-terminal) Plant Hara-Nishimura species et al.,(2004) apoplast Proteinase inhibitor Tobacco Denecke II Calreticulin etal., (1990) Borisjuk et al., (1998) peroxisome SKL, SQL, -SML, -SSL, -Tobacco Kragler SAL (all C-terminal) et al., (1998) vacuole NTPP(N-terminal) Plant Marty, (1999) CTPP (C-terminal) species

Example 5 Generation of Vectors for Transfection of Dicot and MonocotProtoplasts Generation of Vectors for Transfection of Dicot Protoplasts

An expression vector was generated for transient expression ofAngiogenin in dicot protoplast cells. The nucleotide sequence of theexpression cassette contains the ANG gene with an ER signal retentionpeptide regulated by the AtRbcS light regulated promoter and nopalinesynthase (nos) terminator from Agrobacterium tumefaciens foraccumulation in dicot plant tissue (1031312_AtRbcS-p_ANG_nos-t; FIGS. 11and 12).

A control vector (0957286 CaMV35s-p_turboGFP_nos-t; FIG. 13) encoding acassette for expressing a fluorescent marker (turboGFP) in dicot plantcells was also used to confirm protein expression. The cassette consistsof the CaMV35S promoter, coding sequence for the turboGFP protein whichwas codon-optimised for expression in dicots and the nopaline synthase(nos) terminator.

Generation of Vectors for Transfection in Monocot Protoplasts

An expression vector was generated for transient expression ofAngiogenin in monocot protoplast cells. The nucleotide sequence of theexpression cassette contains the ANG gene with an ER signal retentionpeptide regulated by the TaRbcS light regulated promoter and nopalinesynthase (nos) terminator from Agrobacterium tumefaciens foraccumulation in monocot plant tissue (1031308_TaRbcS-p_ANG_nos-t; FIGS.14 and 15).

A control vector (0957284 ZmUbi-p_dsRED_nos-t; FIG. 16) encoding acassette for expressing a fluorescent marker (dsRED) in monocot plantcells was also used to confirm protein expression. The cassette consistsof the Ubiquitin promoter from Zea mays, coding sequence for the dsREDprotein which is codon-optimised for expression in wheat, and thenopaline synthase (nos) terminator.

Example 6 Generation of Vectors for Stable Transformation and Productionof Transgenic Plants

Expression of the recombinant protein in edible tissue for feed stock orhuman consumption offers a convenient and inexpensive source ofdelivery. However, an added value may also be obtained by the extractionof a recombinant protein as a by-product from the primary source.Accordingly, the combination of elements chosen to regulate theexpression, and direct the angiogenin protein, is central to both thesemethods.

Production of Expression Vectors for Biolistic andAgrobacterium-Mediated Transformation

Base transformation vectors are required to contain all the necessaryelements for bilolistic and Agrobacterium mediated transformation ofplants. To this end, various selectable marker cassettes, containing aselectable marker gene controlled by promoter and terminator regulatorysequences, are required for selection within different transformationprocess, and for distinct plant types.

Expression vectors are generated for biolistic and Agrobacteriummediated transformation by the introduction of expression cassettes,containing the ANG gene with a modified signal sequence driven bytargeted expression promoters, into different base vectors. Expressioncassette promoters and signal sequences will be optimised to aparticular strategy such that the strength and targeted delivery of theprotein will be suited to the final processing of the transgenic plant.

Expression Cassette Containing an ER Signal Peptide and Light RegulatedPromoter for Accumulation in Dicot Plant Tissue

To achieve high levels of protein accumulation in photosynthetic dicotplant tissue a light-regulated promoter (AtRbcS) was combined with theFIG. 3 modified ANG gene containing the KDEL ER retention signal, andthe cauliflower mosaic virus CamV35S terminator sequence (FIG. 17).

An expression vector was generated for stable expression of Angiogeninin dicot cells. The nucleotide sequence of the expression cassettecontains the ANG gene with an ER signal retention peptide regulated bythe AtRbcS light regulated promoter and nopaline synthase (nos)terminator from Agrobacterium tumefaciens for accumulation in dicotplant tissue (pPFG000023 AtRbcS-p_ANG_nos-t; FIGS. 11 and 18).

Expression Cassette Containing an ER Signal Peptide and Light RegulatedPromoter for Accumulation in Monocot Plant Tissue

To achieve high levels of protein accumulation in photosynthetic monocotplant tissue light-regulated promoters (TaRbcS and LpRbcS) were combinedwith the FIG. 6 modified ANG gene containing the KDEL ER retentionsignal, and the cauliflower mosaic virus CamV35S terminator sequence(FIGS. 20 and 21 respectively).

Expression Cassette Containing a ER Signal Peptide and Brassica napusNapin Gene Promoter for Accumulation in Dicot Plant Seed

Recombinant seed offers the possibility of direct use as edible planttissue or is a promising target for extraction. To achieve high levelsof protein accumulation in dicot plant seed, a seed specific promoter(Brassica napus napin gene) was combined with, the FIG. 7 modified ANGgene containing a KDEL ER retention signal, and the cauliflower mosaicvirus CaMV35S or nos terminator sequences (FIGS. 22, 23 and 24).

Expression Cassette Containing a ER Signal Peptide and Zea mays ZeinGene Promoter for Accumulation in Monocot Plant Seed

Recombinant seed offers the possibility of direct use as edible planttissue or is a promising target for extraction. To achieve high levelsof protein accumulation in plant monocot seed, a seed specific promoter(Zea mays zein gene) was combined with, the FIG. 6 modified ANG genecontaining a KDEL ER retention signal, and the cauliflower mosaic virusCaMV35S or nos terminator sequences (FIGS. 25, 26 and 27).

Expression Cassette Containing an Apoplast Signal Peptide andConstitutive Promoter for Secretion in Guttation Fluid

Targeted secretion has the potential of increasing the efficiency ofrecombinant protein production technology by increasing yield,abolishing extraction and simplifying its downstream process. Forexample, by using endoplasmic reticulum signal peptides fused torecombinant protein sequences plants may secrete the protein through theleaf intracellular space into guttation fluid. Guttation is liquidformation at the edges of plant leaves produced at night due to excesswater potential. Guttation fluid can be collected throughout a plant'slife, thus providing a continuous and non-destructive system forrecombinant protein production.

To achieve high levels of protein secretion through guttation in bothmonocots and dicots, the cauliflower mosaic virus (CaMV35S) constitutivepromoter and terminator sequences were combined with, the FIG. 7modified ANG gene containing a tobacco calreticulin apoplast signalpeptide (FIG. 32).

Expression Cassette Containing an Apoplast Signal Peptide andRoot-Specific Promoter for Rhizosecretion in Dicots

Targeted and directed expression can be also used to generaterhizosecretion, a method for the production and secretion of recombinantproteins from roots (Gleba, et al., 1998). Expression of ANG using anArabidopsis root specific promoter (AtPHTI) and targeted by a tobaccocalreticulin apoplast signal peptide the recombinant protein could beextracted from rhizosecretion of hydroponically grown transgenic monocotplants (FIG. 33).

Expression Cassette Containing an Apoplast Signal Peptide andRoot-Specific Promoter for Rhizosecretion in Dicots

Targeted and directed expression can be also used to generaterhizosecretion, a method for the production and secretion of recombinantproteins from roots (Gleba, et al., 1998). Expression of ANG using aHordeum vulgare root specific promoter (HvPHTI) and targeted by atobacco calreticulin apoplast signal peptide the recombinant proteincould be extracted from rhizosecretion of hydroponically growntransgenic plants (FIG. 34).

Expression Cassette Containing an Oleosin Promoter and ANG_OleosinFusion Gene for Extraction from Oil Bodies

To achieve high levels of protein in oil bodies, the Arabidopsis oleosinpromoter and CaMV35S terminator were combined with, the oleosin_ANGfusion gene (FIG. 9) to produce an expression cassette (FIG. 35).

Expression Cassette for Transformation of the Plant Chloroplast Genome

Many biopharmaceutical transgenes have been stably integrated andexpressed using the tobacco chloroplast genome to confer desiredagronomic traits or express high levels of protein (Daniell et al.,2005). The FIG. 7 modified ANG gene has been paired with the tobacco16sRNA operon (Prrn) promoter and terminator sequences (Zoubenko, etal., 1994) to express the angiogenin gene in chloroplasts (FIG. 36).

Example 7 Production of Transgenic Plants Expressing Chimeric AngiogeninGenes

The genetic constructs may be introduced into the plant by any suitabletechnique. Techniques for incorporating the genetic constructs of thepresent invention into plant cells (for example by transduction,transfection or transformation). Such techniques includeAgrobacterium-mediated introduction, electroporation of tissues, cellsand protoplasts, protoplast fusion, injection into reproductive organs,injection into immature embryos and high velocity projectileintroduction to cells, tissues, calli, immature and mature embryos,biolistic transformation and combinations thereof. The choice oftechnique will depend largely on the type of plant to be transformed andthe appropriate vector for the method chosen will be used.

Cells incorporating the genetic constructs of the present invention maybe selected, as directed by the vectors used, and then cultured in anappropriate medium to regenerate transformed plants, using techniqueswell established. The resulting plants may be reproduced, eithersexually or asexually, to produce successive generations of transformedplants.

The present invention may be applied to a variety of plants, includingmonocotyledons [such as grasses (e.g. forage grasses including perennialryegrass, tall fescue, Italian ryegrass, brachiaria, paspalum), sorghum,sugarcane, corn, oat, wheat, rice and barley)], dicotyledons [such asforage legumes (e.g. white clover, red clover, subterranean clover,alfalfa), soybean, lupin, peas, lentils, chickpeas, canola, vegetablebrassicas, lettuce, spinach, fruiting plants (e.g. bananas, citrus,strawberries, apples), oil palm, linseed, cottonseed, safflower,tobacco] and gymnosperms.

Example 8 Transfection of Dicot and Monocot Protoplasts Dicot ProtoplastTransfection of Angiogenin

Protoplasts were released from mesophyll tissue of the dicot, Nicotianatabacum using the method described in Spangenberg and Potrykus, 1996.The viability of tobacco protoplasts was assessed using Evans Blue stainas described in Huang et al., 1986 (FIG. 37).

DNA from two plasmids encoding either an expression cassette designed toexpress the ANG protein under control of the AtRbcS promoter(1031312_TaRbcS-p_ANG_nos-t; FIGS. 11 and 12), or a control expressioncassette designed to express the fluorescent reporter (turboGFP) undercontrol of the constitutive CaMV35S promoter (0957286CaMV35s-p_turboGFP_nos-t; FIG. 13), were purified.

Both plasmid vectors were linearised by restriction endonucleasedigestion and delivered to aliquots of protoplasts cells. After 24hours, successful delivery and gene expression were confirmed byvisualisation of the fluorescent marker in the control samples (FIG.38).

Transient Gene Expression and Detection of Angiogenin in DicotProtoplasts

To detect expression of Angiogenin, DNA-free RNA was purified fromprotoplast samples. Complimentary DNA (cDNA) was synthesised and reversetranscriptase-PCR (RT-PCR) analysis of each sample was conducted usingprimers, as outlined in Table 3, to Angiogenin (ANG F and R) and theendogenous house-keeping gene, Actin (Actin F and R).

TABLE 3 Primers for detection of ANG transgeneand endogenous Actin expression. SEQ ID Primer Name Primer Sequence NO:ANG_Forward (F) 5′ GAACGACATCAAGGCT 45 ATCTG 3′ ANG_Reverse (R) 5′AGCACCGTATCTACAA 46 GGAG 3′ Actin_Forward 5′ CCCTCCCACATGCTA 47 (F)TTCT 3′ Actin_Reverse 5′ AGAGCCTCCAATCCAGA 48 (R) CA 3′ oligo-dT_Reverse5′ TTCTAGAATTCAGCGGCCG 49 (R) CT₃₀RN 3′ poly-T_Reverse 5′TTCTAGAATTCAGCGGCC 50 (R) GCT 3′

Each PCR sample was loaded onto an agarose gel, subjected toelectrophoresis and the DNA was visualised (FIG. 39).

The integrity of the cDNA of both turboGFP and ANG transfectedprotoplast samples was confirmed by the presence of a band of expectedsize (524 bp) from samples amplified with the Actin primers. (FIG. 39,lanes 9 and 11, respectively). Confirmation that product amplificationdoes not occur from the transfected DNA template can be observed by theabsence of a band from both turboGFP and ANG transfected protoplastsamples amplified with the same primers to which noreverse-transcriptase was added (FIG. 39, lanes 8 and 10).

Expression of Angiogenin was confirmed by the presence of a band ofexpected size (138 bp) in samples amplified with primers to ANG fromcells transfected with 1031308 AtRbcS-p_ANG_nos-t (FIG. 29, lane 4) andthe absence of a band in samples with the same primers to which noreverse-transcriptase was added (FIG. 39, lane 3). A positive controlperformed with ANG primers and 1031308 AtRbcS-p_ANG_nos-t plasmid DNA isobserved in FIG. 39, lane 6 and indicates the size of the expectedfragment.

Monocot Protoplast Transfection of Angiogenin

Protoplasts were released from mesophyll tissue of the monocot, Triticumaestivum using the method described in Spangenberg and Potrykus, 1996.The viability of tobacco protoplasts was assessed using Evans Blue stainas described in Huang et al, 1986 (FIG. 40).

DNA from two plasmids encoding either an expression cassette designed toexpress the ANG protein under control of the TaRbcS promoter (1031308AtRbcS-p_ANG_nos-t; FIGS. 14 and 15) or a control expression cassettedesigned to express the fluorescent reporter (dsRED) under control ofthe constitutive ubiquitin promoter from Zea mays (0957284ZmUbi-p_dsRED_nos-t; FIG. 16), were purified. Plasmid DNA was deliveredto aliquots of protoplasts cells. After 24 hours, successful deliveryand gene expression were confirmed by visualisation of the fluorescentmarker in the control samples (FIG. 41).

Transient Gene Expression and Detection of Angiogenin in MonocotProtoplasts

To detect expression of Angiogenin, DNA-free RNA was purified fromprotoplasts and cDNA was synthesised with a oligo-dT reverse primer(Table 3). RT-PCR analysis of each sample was conducted using forwardprimers designed to Angiogenin or Actin and a poly-T reverse primer(Table 3) designed to anneal to the adapter sequence of the oligo-dTprimer from which cDNA was synthesised, ensuring that there was noamplification from plasmid template.

Each PCR sample was loaded onto an agarose gel, subjected toelectrophoresis and the DNA was visualised (FIG. 42).

The integrity of the cDNA of all wheat protoplast samples was confirmedby the presence of a band of expected size (920 bp) from samplesamplified with the Actin_F and poly-T_R primer. (FIG. 42, lanes 8 and10) and absence of a band from samples amplified with the same primersto which no reverse-transcriptase was added (FIG. 42, lanes 7 and 9).

Expression of Angiogenin (Rnase5) was confirmed by the presence of aband of expected size (740 bp) in samples amplified with primers toANG_F and poly-T_R primer from cells transfected with 1031312TaRbcS-p_ANG_nos-t (FIG. 42, lane 4) and the absence of a band insamples with the same primers to which no reverse-transcriptase wasadded (FIG. 42, lane 3) and from samples that were not transfected with1031312 TaRbcS-p_ANG_nos-t (FIG. 42, lanes 1 and 2).

Example 9

Agrobacterium-Mediated Transformation of Canola (Brassica napus) forExpression of Chimeric Angiogenin Genes

Binary vectors containing chimeric ANG genes under control of differentpromoters are used for Agrobacterium-mediated transformation of Brassicanapus hypocotyl segments as outlined below and demonstrated in FIG. 43.

Brassica napus seeds are surface sterilised in 70% ethanol for 2minutes, washed 3 times in sterile water then further surface sterilisedin a solution containing 1% (w/v) Calcium hypochlorite and 0.1% (v/v)Tween 20 for 30 minutes. The seeds are washed at least 3 times insterile water and planted in 120 ml culture vessels containing asolidified germination medium containing 1× Murashige and Skoog(Murashige and Skoog Physiol. Plant, 15: 473-497, 1962) macronutrients,1× micronutrients and B5 organic vitamins, supplemented with 500 mg/LMES, 2% (w/v) sucrose at a pH of 5.8 with the addition of 4 g/L Gelrite.The vessels are incubated at 25° C. under 16 h light/8 h dark conditionsfor 7 days to encourage germination.

After 7 days, seedlings of Brassica napus (whole seedlings) aretransferred to a liquid medium consisting of 1× Murashige and Skoogmacronutrients, 1× micronutrients and B5 organic vitamins, supplementedwith 500 mg/L MES, 3% (w/v) sucrose at a pH of 5.8. Seedlings aregrouped together and the roots and cotyledons removed prior to cuttingthe hypocotyls into 7-10 mm sections and plating on 9×1.5 cm petridishes containing a preconditioning medium consisting of 1× Murashigeand Skoog macronutrients, 1× micronutrients and B5 organic vitamins,supplemented with 500 mg/L MES, 3% (w/v) sucrose at a pH of 5.8solidified with 6.4 g/l Bacto-Agar.

Hypocotyl sections are cultured for 24 hours prior to inoculation withan Agrobacterium suspension OD₆₀₀=0.2 for 30 minutes consisting of 1×Murashige and Skoog macronutrients, 1× micronutrients and B5 organicvitamins, supplemented with 500 mg/L MES, 100 μM Acetosyringone, 3%(w/v) sucrose at a pH of 5.8.

Following inoculation, hypocotyl sections are blotted on sterile papertowels and transferred to 9×1.5 cm petri dishes containing 1× Murashigeand Skoog macronutrients, 1× micronutrients and B5 organic vitamins,supplemented with 500 mg/L MES, 100 μM Acetosyringone, 1 mg/L 2,4-D, 3%(w/v) sucrose at a pH of 5.8 solidified with 8 g/l Bacto-Agar. Explantsare incubated at 25° C. under 16 h light/8 h dark conditions for 72hours for co-cultivation.

Following co-cultivation, 20-30 hypocotyl explants are transferred to9×1.5 cm petri dishes containing a solidified selection mediumconsisting of 1× Murashige and Skoog macronutrients, 1× micronutrientsand B5 organic vitamins, supplemented with 500 mg/L MES, 1 mg/L 2,4-D,3% (w/v) sucrose at a pH of 5.8 solidified with 8 g/l Bacto-Agar,supplemented with 250 mg/l timentin and 10 mg/l hygromycin to select forhygromycin-resistant shoots. Plates are incubated at 25° C. under 16 hlight/8 h dark conditions.

After 7 days hypocotyl explants are transferred to 9×2.0 cm petri dishescontaining a solidified regeneration media consisting of 1× Murashigeand Skoog macronutrients, 1× micronutrients and B5 organic vitamins,supplemented with 500 mg/L MES, 1 mg/L 2,4-D, 3% (w/v) sucrose at a pHof 5.8 solidified with 8 g/l Bacto-Agar, supplemented with 4 mg/l BAP, 2mg/l Zeatin, 5 mg/l Silver Nitrate, 250 mg/l timentin and 10 mg/lhygromycin. Plates are incubated under direct light at 25° C. underfluorescent light conditions (16 hr light/8 hr dark photoperiod; 55 μmolm⁻² sec⁻¹) for 4 weeks to encourage shoot development.

Regeneration is monitored weekly and hypocotyl explants transferred tofresh 9×2.0 cm petri dishes containing solidified regeneration media, RMsupplemented with 4 mg/l benzyladenine, 2 mg/l zeatin, 5 mg/l silvernitrate, 250 mg/l timentin and 10 mg/l hygromycin for 6-8 weeks toencourage shoot development.

Hygromycin-resistant (Hyg^(r)) shoots are transferred to 120 ml vesselscontaining solidified root induction medium, RIM1, consisting of 1×Murashige and Skoog macronutrients, 1× micronutrients and B5 organicvitamins, supplemented with 500 mg/L MES, 1 mg/L 2,4-D, 1% (w/v) sucroseat a pH of 5.8 solidified with 8 g/l Bacto-Agar supplemented with 250mg/l timentin. Shoots are incubated under direct fluorescent light at25° C. (16 hr light/8 hr dark photoperiod; 55 μmol m⁻² sec⁻¹) toencourage shoot elongation and root development over 4-5 weeks. AllHyg^(r) shoots with developed shoot and root systems are transferred tosoil and grown under glasshouse conditions.

Example 10

Biolistic Transformation of Wheat (Triticum aestivum L.) for Expressionof Chimeric Angiogenin Genes

Transformation vectors containing chimeric ANG genes are used forbiolistic transformation of wheat (Triticum aestivum L. MPB Bobwhite 26)as outlined below.

Step 1 (Donor Plant Production):

Triticum aestivum (Bobwhite 26) seed is used for the production of donorplant material. Wheat plants are grown in a nursery mix consisting ofcomposted pine bark, perlite and vermiculite, with five plants per potto a maximum pot size of 20 cm. Plants are kept under glasshouseconditions at approximately 22-24° C. for 12-16 weeks (FIG. 45A). Oncethe first spike emerges from the flag leaf, plants are tagged andembryos collected from the tallest heads 12-15 days post anthesis.

Step 2 (Day 1):

Spikes at the desired stage of development are harvested (FIG. 45B).Caryopsis are removed from the spikes and surface sterilised for 20minutes in a 0.8% (v/v) NaOCl solution and rinsed at least four times insterile distilled water.

Embryos up to 10 mm in length are aseptically excised from eachcaryopsis (removing the axis) using a dissecting microscope and culturedaxial side down on an osmotic medium (E3maltose) consisting of 2×Murashige and Skoog (1962) macronutrients, 1× micronutrients and organicvitamins, 40 mg/L thiamine, 150 mg/L L-asparagine, supplemented with 15%(w/v) maltose, 0.8% (w/v) Sigma-agar and 2.5 mg/L 2,4-D (FIGS. 45C andD). Embryos are cultured on 60 mm×15 mm clear polypropylene petriedishes with 15 mL of media. Culture plates are incubated at 24° C. inthe dark for 4 hours prior to bombardment. Embryos are bombarded using aBioRad PDS1000 gene gun at 900 psi and at 6 cm with 1 μg of vectorplasmid DNA precipitated onto 0.6 μm gold particles. Followingbombardment, embryos are incubated overnight in the dark on the osmoticmedia.

Step 3 (Day 2):

Embryos are transferred to a callus induction medium (E3calli)consisting of 2× Murashige and Skoog (1962) macronutrients and 1×micronutrients and organic vitamins, 40 mg/L thiamine, 150 mg/LL-asparagine, supplemented with 6% (w/v) sucrose, 0.8% (w/v) Sigma-agarand 2.5 mg/L 2,4-D. Embryos are cultured for two weeks at 24° C. in thedark.

Step 4 (Day 16):

After 2 weeks of culture on E3calli, embryos have produced embryogeniccallus and are subcultured onto a selection medium (E3Select) consistingof 2× Murashige and Skoog (1962) macronutrients and 1× micronutrientsand organic vitamins, 40 mg/L thiamine, 150 mg/L L-asparagine,supplemented with 2% (w/v) sucrose, 0.8% (w/v) Sigma-agar, 5 mg/L of D,Lphosphinothricin (PPT) and no plant growth regulators (FIGS. 45E-G).Cultures are incubated for further 14 days on E3Select at 24° C. in thelight and a 12-hour photoperiod.

Step 5 (Day 30):

After 14 days culture on E3Select, embryogenic callus is sub-culturedonto fresh E3Select for a further 14 days (FIGS. 45E-G).

Step 6 (Day 44):

After about 4 weeks on E3Select, developing plantlets are excised fromthe embryonic callus mass and grown for a further three weeks in 65mm×80 mm or 65 mm×150 mm polycarbonate tissue culture vessels containingroot induction medium (RM). Root induction medium consists of 1×Murashige and Skoog (1962) macronutrients, micronutrients and organicvitamins, 40 mg/L thiamine, 150 mg/L L-asparagine, supplemented with 2%(w/v) sucrose, 0.8% (w/v) Sigma-agar, and 5 mg/L of PPT (FIG. 45H).Remaining embryogenic callus is sub-cultured onto E3Select for another14 days.

Step 7 (Day 65+):

Regenerated plantlets surviving greater than 3 weeks on RM with healthyroot formation are potted into a nursery mix consisting of peat and sand(1:1) and kept at 22-24° C. with elevated humidity under a nurseryhumidity chamber system. After two weeks, plants are removed from thehumidity chamber and hand watered and liquid fed Aquasol™ weekly untilmaturity. The T₀ plants are sampled for genomic DNA and molecularanalysis. T₁ seed is collected and planted for high-throughput Q-PCRanalysis.

Example 11

Agrobacterium-Mediated Transformation of Wheat (Triticum aestivum L.)for Expression of Chimeric Angiogenin Genes

Agrobacterium-mediated transformation of bread wheat is represented inFIG. 45. Wheat donor plants ready are harvested for use as source ofembryo explants for Agrobacterium mediated transformation. Postinfection from Agrobacterium, callus material is regenerated on tissueculture medium under appropriate selection until regenerating shoots areobserved. Following several rounds of selection the rooted plant ispotted in soil.

Example 12

Agrobacterium-Mediated Transformation of Tobacco (Nicotiana benthamiana)for Expression of Chimeric Angiogenin Genes

In tobacco Agrobacterium-transformation adventitious shoots can beregenerated at high frequencies from leaf explants.Agrobacterium-mediated tobacco transformation is a four stage process.

1. Inoculation of regenerative explants with a cell suspension ofAgrobacterium.

2. Co-cultivation of inoculated explants on regeneration medium for 2-3days during gene transfer occurs.

3. Regeneration and selection of transformed shoots and the eliminationof bacteria.

4. Biochemical and molecular analysis of putative transgenic plants.

Example 13

Agrobacterium-Mediated Transformation of Alfalfa (Medicago sativa) forExpression of Chimeric Angiogenin Genes

Binary vectors containing chimeric ANG genes under control of differentpromoters are used for Agrobacterium-mediated transformation of Medicagosativa petiole explants from highly-regenerable alfalfa (M. sativa)clones.

Following co-cultivation with Agrobacterium tumefaciens strain LBA 4404harbouring the binary vector, the alfalfa explants were washed withmedium containing cefotaxime and used for induction of embryogeniccallus under selective medium containing 25 mg/l kanamycin. Transgenicembryogenic alfalfa calli were recovered and allowed to regeneratetransgenic alfalfa shoots, which were transferred on rooting mediumleading to the recovery of transgenic alfalfa plants expressing chimericANG genes.

Example 14

Biolistic Transformation of Perennial Ryegrass (Lolium perenne) forExpression of Chimeric Angiogenin Genes

Biolistic co-transformation of perennial ryegrass with the vectorscontaining the TaRbcS and LpRbcS regulatory sequences, driving theexpression of the ANG gene (FIGS. 19, 20 and 21) and the pACH1 vectorfor hygromycin resistance is conducted on embryogenic calli forperennial ryegrass. The pACH1 vector was previously constructed and hasbeen used successfully in plant transformation experiments (Bilang, etal., 1991; Spangenberg, et al., 1995a; Spangenberg, et al., 1995b; Ye,et al., 1997; Bai, et al., 2001). The perennial ryegrass biolistictransformation method is outlined in FIG. 44.

Example 15

Agrobacterium-Mediated Transformation of White Clover (Trifolium repens)for Expression of Chimeric Angiogenin Genes

Vectors containing chimeric ANG genes under control of differentpromoters are used for Agrobacterium-mediated transformation ofTrifolium repens cotelydons as outlined below.

All material in tissue culture are grown at 24° C. with a 16 h light/8 hdark regime. White clover seeds are washed in 70% v/v ethanol,surface-sterilised in 1.5% sodium hypochlorite (12.5 g/L activechlorine), rinsed in sterile distilled water and imbibed overnight at 4°C. in the dark. Cotyledonary explants are excised with a 1-2 mm segmentof hypocotyl attached. Explants are incubated in Agrobacterium culture(OD₆₀₀=approx 0.35) for 40 min and co-cultivated on regeneration medium,consisting of: 1× Murashige and Skoog Basal Medium (Sigma), 30 g/Lsucrose, 5 M thidiazuron (Sigma), 0.5 M naphthalene-acetic acid, 250mg/L cefotaxime (Claforan, Hoechst) and 8 g/L Bacto-Agar(Becton-Dickinson), pH 5.75, supplemented with 40 mg/L acetosyringone.Explants are co-cultivated for 3 days at 24° C., and transferred toregeneration medium containing an appropriate selective agent and aresubcultured every 2-3 weeks. Regenerated shoots are transferred toroot-inducing medium, consisting of: 1×MS basal medium, 15 g/L sucrose,1.2 M indole-butyric acid, 250 mg/L cefotaxime, an appropriate selectiveagent and 8 g/L Bacto-Agar, pH 5.75. Antibiotic-resistant plantlets aretransferred to soil and established under glasshouse conditions. Thewhite clover Agrobacterium-mediated transformation method isdemonstrated in FIG. 46.

Example 16 Production of Transgenic White Clover Plants ExpressingChimeric Angiogenin Genes

Use of constructs containing a light regulated promoter and endoplasmicreticulum retention signal

The AtRbcS_ANG_(—)35S expression cassette was incorporated into a vectorbackbone containing a selectable marker cassette of the neomycinphosphotransferase (npt II) gene driven by the nopaline synthase (nos)promoter and terminator sequences (FIG. 47). This vector was insertedinto the white clover genome by Agrobacterium mediated transformation.

Example 17 Characterisation of Transgenic White Clover Plants ExpressingChimeric Angiogenin Genes Detection of Angiogenin Expression inTransgenic White Clover

Total RNA was extracted from leaves and stems of white clovertransformed with AtRbcS_ANG_(—)35S and an untransformed control. Reversetranscriptase polymerase chain reaction (RT-PCR) analysis was performedusing primers specific to the angiogenin gene to detect angiogeninexpression in the transgenic plants (FIG. 48).

Detection of the Angiogenin Protein in Transgenic White Clover

Total protein extract was obtained from non-transgenic (control) andtransgenic white clover plant tissue and separated using 2DE gelapparatus. Two dimensional protein profiles were compared between thenon-transgenic and transgenic plants. The transgenic tissue was observedto contain a rich protein spot not observed in the control material(FIG. 49). The MOWSE scoring algorithm was used to determine theidentity of this rich protein spot in the transgenic white leaf clover.This was achieved as follows.

The protein spot of interest was excised from the 2DE gel and digestedwith overnight porcine trypsin. The digested protein sample was then C18zip-tipped and spotted onto an MALDI-TOF/TOF mass spectrometry target.The spotted protein sample was then sequenced using MALDI-TOF/TOF massspectrometry (FIG. 50). The observed peptide masses obtained from thepeptide mass fingerprint data and the observed peptide ion fragmentationmasses obtained from the peptide ion fragmentation pattern were thencombined together and searched against the NCBInr sequence database ofknown calculated peptide masses and known calculated ion fragmentationmasses. The mass spectra obtained by MALDI-TOF/TOF mass spectrometrymatched bovine angiogenin in the NCBInr sequence database. The proteinscore and ion scores received were positive for bovine angiogenin usingthe MOWSE scoring algorithm.

Protein Quantification of Bovine Angiogenin in Soluble Transgenic WhiteLeaf Clover Extract

Approximately, 50 ug of the soluble transgenic white leaf clover extractwas loaded on to the 2DE gel. Bovine angiogenin represents 10% of thesoluble transgenic white leaf clover extract and is therefore 5 mg ofthe soluble transgenic white leaf clover extract. This was determined bydensitometry using Progenesis PG240 software (Non Linear Dynamics,Newcastle upon Tyne, UK).

The soluble transgenic white leaf clover extract was prepared byhomogenising 200 mg of ground plant tissue in 1.5 ml of homogenistionbuffer. The level of expression in transgenic white leaf clover equatesto 7.5 mg of bovine angiogenin per milligram of plant extract. Thislevel is equivalent to angiogenin expression in bovine cows milk whichis between 4-8 mg/ml.

Example 18 Production of Transgenic Plants Co-Expressing Angiogenin andOther Proteins for Enhanced Angiogenin Productivity

It would be possible to pyramid existing technologies to generate asignificant impact on the efficacy of a variety of applications byincreasing the range of productivity in plants.

The productivity of angiogenin expressed in plants may be enhancedthrough co-expression with antimicrobials, protease inhibitors, RNaseinhibitors, follistatin or delayed plant organ senescence nucleic acidsand constructs.

Technologies for the extend life of plants (patent PCT/AU01/01092),increased biomass and high fructans (patent PCT/AU2009/001211), havebeen established. Pyramiding the current application technology withtechnologies that address these other factors should greatly increaseplant health and production which should, in turn, increase animalhealth and production, as well as enhance the generation of value addedproducts in plant biomass.

Example 19

Agrobacterium-Mediated Transformation of Arabidopsis (Arabidopsisthaliana) for Expression of Chimeric Angiogenin Genes

Vectors containing chimeric ANG genes under control of differentpromoters are used for Agrobacterium-mediated transformation ofArabidopsis thaliana as outlined below.

1. Inoculation with a cell suspension of Agrobacterium to Arabidopsisusing infiltration to facilitate access of Agrobacterium to immatureflowers where T-DNA transfer may then take place.

2. Plant growth and monitoring and collection of potentially transgenicseed.

3. Regeneration and selection of transformed seeds on germination mediawith appropriate selection antibiotic.

4. Biochemical and molecular analysis of putative transgenic plants.

Example 20 Production of Transgenic Arabidopsis Plants ContainingChimeric Angiogenin Genes Use of Constructs Containing a Light RegulatedPromoter and Endoplasmic Reticulum Retention Signal

The AtRbcS_ANG_nos expression cassette (FIG. 11) was incorporated into avector backbone containing a selectable marker cassette of the hygromcinphsophotransferase (hptII) gene driven by the CSVMV promoter and CaMV35Sterminator sequences (FIG. 18). This vector was inserted into theArabidopsis genome by Agrobacterium mediated transformation.

Example 21 Characterisation of Transgenic White Clover Plants ContainingChimeric Angiogenin Genes Detection of the Angiogenin Gene in TransgenicArabidopsis

DNA was extracted from Arabidopsis leaves of two different transgeniclines with AtRbcS_ANG_nos and a wild-type untransformed control.Polymerase chain reaction (PCR) analysis was performed using primersspecific to the angiogenin gene (Table 3) to detect the presence of theangiogenin gene in the transgenic plant lines (FIG. 51).

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1-16. (canceled)
 17. A plant cell, said plant cell containing (a) anucleic acid encoding an angiogenin, and/or (b) an angiogeninpolypeptide, wherein the plant cell is optionally part of a plantcallus, plant, seed or other plant part.
 18. The plant cell according toclaim 17, wherein said cell contains (a) a nucleic acid encoding anangiogenin of SEQ ID NO: 2, or a functionally active fragment or variantof SEQ ID No. 2, and/or (b) an angiogenin polypeptide of SEQ ID NO: 2,or a functionally active fragment or variant of SEQ ID No.
 2. 19. Aplant-produced angiogenin.
 20. A food, beverage, food supplement,nutraceutical, pharmaceutical, feedstock, or veterinary productcomprising a plant-produced angiogenin.
 21. A method of producing atransformed plant cell expressing an angiogenin sequence, said methodcomprising introducing a nucleic acid encoding an angiogenin into aplant cell to produce a transformed plant cell; and culturing thetransformed plant cell to produce a transformed plant cell expressingthe nucleic acid encoding an angiogenin.
 22. The method according toclaim 21 wherein the nucleic acid encoding an angiogenin encodes apolypeptide comprising SEQ ID NO: 2, or a functionally active fragmentor variant thereof.
 23. The method of claim 21, wherein the nucleic acidencoding an angiogenin is a sequence from which an N-terminal signalsequence has been removed.
 24. The method of claim 23, wherein thenucleic acid encoding an angiogenin further encodes a plant signalpeptide in place of the removed N-terminal signal sequence.
 25. Themethod of claim 23, wherein the nucleic acid encoding an angiogeninencodes a polypeptide of Seq ID No. 2 from which the signal peptidesequence has been removed.
 26. The method of claim 25, wherein thenucleic acid encoding an angiogenin further encodes a plant signalpeptide in place of the removed N-terminal signal sequence.
 27. Themethod according to claim 21, wherein the transformed plant cell is partof a plant callus, plant, seed or other plant part.
 28. The method ofclaim 21, wherein the nucleic acid encoding an angiogenin is a variantof an animal angiogenin sequence modified to provide a plant codon bias.29. A method of producing an angiogenin in a plant, said methodcomprising introducing a nucleic acid encoding an angiogenin into aplant cell to produce a transformed plant cell; culturing thetransformed plant cell to produce a transformed plant cell expressingthe nucleic acid encoding an angiogenin; and isolating the angiogeninproduced by the plant cell.
 30. The method according to claim 29 whereinthe nucleic acid encoding an angiogenin encodes a polypeptide comprisingSEQ ID NO: 2, or a functionally active fragment or variant thereof. 31.The method according to claim 14 wherein the nucleic acid encoding anangiogenin is a sequence from which an N-terminal signal peptide hasbeen removed.
 32. The method according to claim 31 wherein the nucleicacid encoding an angiogenin further encodes a plant signal peptide inplace of the removed N-terminal signal peptide.
 33. The method of claim29, wherein the nucleic acid encoding an angiogenin is a sequence fromwhich an N-terminal signal peptide sequence has been removed.
 34. Themethod of claim 33, wherein nucleic acid encoding an angiogenin furtherencodes a plant signal peptide in place of the removed N-terminal signalsequence.
 35. The method according to claim 29, wherein the transformedplant cell is part of a plant callus, plant, seed or other plant part36. The method of claim 29, wherein the nucleic acid encoding anangiogenin is a variant of an animal gene modified to provide a plantcodon bias.
 37. An artificial construct comprising a nucleic acidencoding an angiogenin and a promoter, operatively linked to the nucleicacid encoding an angiogenin, wherein the promoter is effective forenabling expression of the nucleic acid encoding an angiogenin in aplant cell.
 38. The artificial construct of claim 37, further comprisinga nucleic acid sequence encoding a mediator or modulator of angiogeninactivity.
 39. The artificial construct of claim 37, wherein the nucleicacid encoding an angiogenin comprises a sequence made from an animalangiogenin sequence by codon usage adaptation for expression in plants.40. A nucleic acid encoding an angiogenin, wherein the nucleic acidcomprises a sequence made from an animal angiogenin sequence by codonusage adaptation for plants to enable expression of said nucleic acidencoding an angiogenin in a plant cell.
 41. A method of enhancingexpression, activity or isolation of angiogenin in plants, said methodcomprising co-expressing angiogenin with a mediator or modulator ofangiogenin activity in a plant cell.
 42. A chimeric nucleic acidsequence comprising a nucleic acid encoding an angiogenin and a plantsignal peptide.
 43. The chimeric nucleic acid sequence of claim 42,wherein the nucleic acid encoding an angiogenin comprises a sequencemade from an animal angiogenin sequence by codon usage adaptation forexpression in plants.